While the first comic shared enough discrepancies with the games to suggest it was not canon to the series, the second comic, Aquilus was confirmed by Ubisoft to be part of the Assassin's Creed universe, both in the Universe video and in the Encyclopedia. However, the third comic, Accipiter did not seem to be part of the canon; while it had some similarities to Assassin's Creed: Brotherhood it was a completely original story with no references to Ezio Auditore da Firenze's time in Rome, and with the modern-dayAssassins searching for the Ankh instead of Ezio's Apple of Eden.

A major contradiction of the first comic was that Subject 16 was alive. Official guidebooks for Assassin's Creed stated that Abstergo's doctors "were too late to save him" after he cut a vein to produce the messages seen by Desmond Miles through Eagle Vision and it was explicitly stated by Alexandre Amancio that Subject 16 was physically dead. Lucy Stillman appeared to act uncaring towards him and was irritated by his presence later in the first comic, which contradicted greatly to the visible guilt and grief she showed about him during the games. While the original French edition of the first volume gave his name as "Michael," the English translation changed it to "Clay."

Another point that differed from the games was that the Apples of Eden were called "Spheres."

The drawings shown in Desmond's room bore no resemblance to those of the game.

The progression into Assassin's Creed II's main events via the modern-day Assassins and Animus greatly differed, some characters being entirely omitted as of the first comic.

There was also a contradiction in that Aquilus' memories were relived after his child's conception, even to the point of his own death — something that genetic memory constraints should not allow.

Lucy is depicted as a very different character in this series than she is in the games. For instance, she showed a much colder side rather than the calm and empathetic personality that she displayed in the games - to the point that Desmond jokingly called her "Ice Queen", although she did lighten up as the series progressed, developing into a character much closer to that of the games. Her feelings for Desmond were also less ambiguous than they were in the games, with the two actually kissing at one point. Lucy was also shown to not be a Templar double agent like in the games and was, in fact, nearly killed while trying to protect Desmond when the Templars found them in Monteriggioni.